Metastasis or spread of cancer cells to locations other than the primary location is the primary cause of death due to cancer. The mechanism of metastasis is not well understood and there are very few drugs or inhibitors that block the process effectively. Most drugs or other therapies for treating cancer are aimed at killing the cancer cells and often kill many normal cells leading to adverse side effects. Some drugs are designed to block the growth of tumors or their blood supply (e.g., inhibit angiogenesis), but these drugs are often marginally effective and interfere with normal physiologic functions, again leading to adverse side effects.
Epithelial-to-mesenchymal transition (EMT) for cells occurs during normal development and at the beginning of epithelial cancers in which the cells acquire an invasive or migratory phenotype (Liu, Radisky et al. 2005; Turley, Veiseh et al. 2008). This change is characterized by the loss of cell-cell adhesion and cytoskeletal rearrangement (Thiery and Sleeman, 2006). Epithelial cell-cell adhesion is predominantly maintained by the apical junction complex (AJC), which is formed by tight junctions (TJs) and adherens junctions (AJs) (Troyanovsky, Klingelhofer et al. 1999). Tight Junctions (TJs) are formed when integral membrane proteins (e.g., the claudin family of proteins and occludins) and junctional adhesion molecules (JAMs) interact with adaptor proteins of the zonula occludens (ZO) family (e.g., ZO-1, ZO-2 and ZO-3) to link TJs with the actin cytoskeleton (Shin, Fogg et al. 2006; Fanning and Anderson 2009). TJs are deregulated in EMT. TJ proteins have been reported to exert major influence over mechanisms that regulate cell polarity, differentiation and migration; and all these processes are central to cancer progression (Brennan et al.; Martin and Jiang, 2009)
Protein Kinase A (PKA), a serine/threnoine kinase, is activated by increased levels of cyclic AMP (cAMP) in localized microdomains and catalyzes key cellular processes such as metabolism, gene transcription, ion channel conductivity, cell growth, cell division and actin cytoskeleton rearrangements (Carr, Stofko-Hahn et al. 1991; Francis and Corbin 1994). Given the broad spectrum of signaling events controlled by PKA, the fidelity of PKA action is tightly regulated by spatial mechanisms that target PKA toward its substrate and by temporal mechanisms involving phosphodiesterases that degrade cAMP to limit the duration of the cellular effects of PKA (Wong and Scott 2004; Lynch, Baillie et al. 2005). Zonula occludens-1 (ZO-1), a scaffolding protein with multiple interaction domains, has been shown to recruit protein kinases, phosphatases, small guanine triphosphatases (GTPases), and transcription factors to the TJ complex (Gonzalez-Mariscal, Tapia et al. 2008). This leads to the juxtaposition of structural (actin and spectrin) and regulatory (actin-binding proteins, GTPases, kinases) proteins with transmembrane proteins (Gonzalez-Mariscal, Betanzos et al. 2000; Kohler and Zahraoui 2005), facilitating interactions among them. PKA signaling regulates both the assembly and opening of the paracellular route in epithelial and endothelial cells (Gonzalez-Mariscal, Tapia et al. 2008). Although PKA seems to enhance TJ assembly and barrier function in normal epithelial cells, PKA has been shown to activate RhoA, a founding member of the small Rho GTPase family, at the apical side of the cell to promote TJ disassembly, increased paracellular permeability, loss of TJ functionality, and actin disorganization in apical and medial regions of epithelial cancer cells (Leve, de Souza et al. 2008).
Higher TJ protein phosphorylation reduces TJ functionality (Singer, Stevenson et al. 1994). ZO-1 is a member of the membrane-associated guanylate kinase (MAGuK) family of large scaffolding and signaling proteins that share several protein-binding domains including three PDZ domains, a SRC homology domain (SH3), and a GUK domain (Fanning et al., 2002; Fanning and Anderson 2009). ZO-1 is a major scaffolding protein and a key organizer of TJs at the plasma membrane (Fanning et al., 2002; Li et al., 2005). ZO-1 binds directly with occludin, claudins and junction adhesion molecules (JAMs) to form TJs (Anderson and Van Itallie, 2008; Denker and Nigam, 1998); associates with other proteins that regulate epithelial cell polarity and paracellular permeability; and serves as a bridge between cell surface and actin cytoskeleton (Fanning et al., 1998; Hurd et al., 2003; Van Itallie and Anderson, 2004; Zahraoui, 2004).
Calcitonin receptor (CTR) is a member of the class B family of G Protein-coupled receptors (GPCRs), which contain numerous drug targets (Conner et al., 2004; Dong et al., 2004). CTR binds calcitonin (CT) and other ligands of the CT family to maintain calcium homeostasis in the bone and the kidneys (Katafuchi et al., 2009; Purdue et al., 2002). However, CTR expression in multiple organs and its actions on cell growth and differentiation demonstrates that CTR has a more diverse role than just the maintenance of calcium homeostasis (Findlay, 2006; Huang et al., 2006; Shah, 2009). The expression of CT and CTR is up-regulated in metastatic prostate cancer (PC) (Thomas, Chiriva-Internati et al. 2007; Shah 2009; Chien et. al., 2001b). Activation of the autocrine CT-CTR axis increases cell proliferation, chemo-resistance, and invasiveness of prostate cancer cell lines (Ritchie, Thomas et al. 1997; Thomas and Shah 2005; Thomas, Chigurupati et al. 2006; Thomas, Chiriva-Internati et al. 2007; Thomas, Muralidharan et al. 2007). Moreover, CTR destabilizes tight junctions (TJs) as assessed by transelectric epithelial resistance (TER) and paracellular permeability (PCP) of multiple prostate cancer cell lines (Shah et al., 2009). Two small molecules, phenyl-methylene hydrantoin (PMH) and its S-ethyl derivative (S-PMH) were found to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis, and were noted to be potential drug candidates for CT-positive androgen-independent prostate cancers (Shah et al., 2009).
Human CTR (hCTR) exists in three or more isoforms formed by alternative splicing of the same primary transcript (Egerton et al., 1995). Recent studies suggest selective expression of isoform 2 of hCTR (hCTR2) in basal, but not luminal, epithelium of human prostate cells (Chien et al., 2001b; Shah et al., 1994). However, this spatial specificity is lost in malignancy, and the abundance of CTR transcripts is increased with tumor progression. CTR stimulates several processes associated with tumor growth, invasion, angiogenesis and metastasis. CTR serves as an important factor in the progression of a localized prostate cancer to its metastatic form (Chigurupati et al., 2005; Sabbisetti et al., 2005; Shah et al., 2008; Thomas et al., 2006).
hCTR2 lacks an 16-amino acid insert in the first intracellular loop, which enables it to couple to both stimulatory GTP binding protein (Gas) and Gaq. In addition, CTR destabilizes tight and adherens junctions and activates non-G protein-coupled signaling pathways such as phosphoinositide-3-kinase (PI3K)-Akt-survivin and WNT/b-catenin (7,13). (Shah et al., 2009a; Thomas and Shah, 2005). Class B G protein-coupled receptors (GPCRs) may also activate non-G protein-mediated signaling (Chen et al., 2009; Hall et al., 1999). For example, cytoplasmic tails of some GPCRs interact with scaffolding proteins through protein-interacting domains such as SRC homology 2 (SH2) or PSD-95-disc large-zonnula occludens (PDZ) to regulate receptor-mediated signal transduction, receptor regulation, or receptor biosynthesis (Bockaert et al., 2004). The four C-terminal residues of CTR (aa 471-74, E-S-S-A (SEQ ID NO:1) form a canonical type I PDZ domain-binding motif, which may allow it to interact with a PDZ containing scaffolding protein(s) to activate non-G protein-mediated signaling (Bockaert et al., 2003; Tao and Johns, 2004). Other receptors are known that also contain C-terminal PDZ-binding motif, for example, parathyroid hormone (PTH) receptor and adrenergic receptors. Moreover, PTH receptor has been found to be up-regulated in prostate cancer and may be associated with skeletal metastasis. (Mahon, 2009; Lupp et al., 2010; Liao et al., 2006; and Tovar et al., 2002)
Although calcitonin receptor (CTR) activates multiple signaling pathways that may mediate multiple effects of CTR on prostate cancer cells, Gas-activated signaling plays an important role in its proinvasive actions. For example, constitutive activation of Gas mimics CTR-induced increase in metastasizing capacity of prostate cancer cells, and inhibitors of cAMP-dependent protein kinase (PKA) attenuate CTR-mediated invasion of prostate cancer cells (Chien, Wong et al. 1999; Sabbisetti, Chirugupati et al. 2005). However, the precise mechanism by which CTR stimulates prostate cancer metastasis has not been previously identified.
Pancreatic cancer cell lines with activated CT-CTR autocrine axis such as PC-3M and DU-145 cells display greater invasiveness and metastatic potential than cell lines with inactive CT-CTR autocrine axis such as LNCaP and PC-3 cells (Thomas, 2006). LNCaP and PC-3 cells acquire or increase invasive phenotype with activation of CT-CTR autocrine axis (Thomas, 2006). In contrast, PC-3M cells lose their invasive phenotype with knock-down of either CT or CTR expression (Shah et al., 2009; Thomas et al., 2006). Anti-sense CT ribozyme therapy inactivates CT-CTR axis in LPB-Tag transgenic mice, also sharply reducing growth of spontaneously induced prostate tumors (Shah et al., 2008).